Methods and compositions for bone graft implants

ABSTRACT

An autologous platelet gel for bone grafts is comprised of a mixture of platelet-rich-plasma activated by calcium chloride solution, and an aqueous suspension of partially frayed Type I collagen.

FIELD OF THE INVENTION

[0001] The present invention relates to methods and compositions forbone repair and, in particular, an implantable, thrombin-free,autologous platelet gel and matrix that promotes bone augmentation andregeneration.

BACKGROUND OF THE INVENTION

[0002] Fibrin glue, also known as fibrin sealant or fibrin gel, is oneof many materials developed in response to a recognized need forimproved hemostatic agents and sealant (Ref. 1). Platelet-basedimplantable gels have been used extensively as wound healing and boneregeneration agents in preferred substitution for fibrin glues. Fibringlues are a two component system comprising, as a first component,concentrated fibrogen, a fibrin stabilizing factor and fibroconectin.The second component includes thrombin, calcium chloride, and aninhibitor of fibrinolysis. The constituents when combined form a fibringel or clot. Thrombin and the calcium cleave the fibrogen to fibrin inthe coagulation cascade and activate factor XIlI which crosslinks fibrininto an organized clot. The gel may be topically applied or in-vitromolded prior to implantation.

[0003] The fibrogen content is customarily plasma based. Such plasma maybe derived from a variety of sources including random donor orsingle-donor cryoprecipitate or from autologous plasma. Homologoussourcing presents numerous quality control problems. Procured from donorblood, certain patient risks may be encountered including compatibilityproblems, disease transmission dangers, clerical and storage errors.While an autologous cryoprecipitate overcomes some of the above, thedonor blood must be procured substantially in advance of surgery, 3 to 5days or more.

[0004] Autologous platelet gel was developed in further response to theabove. Therein, whole blood is obtained from the patient in thepreoperative period and processed in the operating area. Throughdifferential centrifugation, a fraction representing the platelet stratais separated. This strata or platelet-rich-plasma (PRP) is combined withthrombin and calcium chloride representatively using the technique setforth in Whitman et.al, Ref above. It has also been proposed that suchresultant clots be supplemented with calcium phosphate minerals andother osseoparticulates including autologous bone and marrow materialfor use in oral and maxillofacial surgery.

[0005] Autologous platelet gel differs from fibrin glue in the presenceof a high concentration of platelets and a high concentration of nativefibrogen. The platelets, activated by the thrombin, release factors andform scaffolding for the development of a clot. Two of the growthfactors, platelet derived growth factor (PDGF) and transforming growthfactor-beta (TGF-B) are known to promote wound healing. PDGF is anactivator of collangenase during wound healing allowing reshaping ofcollagen for wound strength. It also is known to be chemotactic formonocytes and macrophages. TGF-B is known to activate fibroblasts toform procollagen resulting in collagen deposition within the wound.

[0006] Autologous platelets gels have gained acceptance in the area ofreconstructive oral surgery in connection with ablative surgery of themaxillofacial region, mandibular reconstruction, surgical repair ofalveolar clefts and associated oral-antral/oral-nasal fistulas, andadjunctive procedures related to the placement of osteointegratedimplants. Such platelet gels have also been used in combination withparticulate cancellous bone and marrow grafts (PCBM) (Ref. 1, Ref. 2).It is reported that such platelet gels with the graft material evidencedsubstantially greater maturation rates and bone density than suchimplants without the platelet- rich-plasma. The PDGF and TGF growthfactors were amplified in this approach and the probable primaryinitiators of the results.

[0007] The action of the thrombin in such autologous platelet gels hasbeen recognized as the primary biological release mechanism of thesegrowth factors as discussed in U.S. Pat. No. 5,165,938 to Knighton.While other biological release agents such as collagen, ADP, andsrotonin have been suggested for activating, the performance of thrombinwas preferred and appears to have been adopted in the art as the agentof choice. The thrombin customarily used in both platelet gels andfibrin systems has been a bovine derivative. To reduce potentialxenographic effects, the bovine thrombin has been used in highlypurified form.

[0008] Notwithstanding the improved results reported with the thrombin-based fibrin and platelet systems, there are numerous reports detailingadverse clinical effects that have been linked potentially to the bovinethrombin. Sosolik et. al. reported a prolongation of thrombin time wasassociated with the presence of anti-bovine thrombin antibodiesfollowing surgical procedures when fibrin glues or bovine thrombinpreparations were applied topically and it was suggested that suchexposure could lead to serious bleeding complications during surgery orthe postoperative period (Ref. 3). Spero concluded that bovine-inducedcoagulopathy may occur following surgical exposure to topical bovinethrombin and may result in both postoperative morbidity and mortality ina subset of patients resulting from topical-induced antibodies toclotting factor V following neurolosurgical procedures (Ref. 4). Cmoliket. al. reports coagulopathy occasioned by bovine thrombin-inducedfactor V deficiency after exposure to bovine thrombin in topicalhemostatic agents during cardiovascular or vascular operation (Ref. 5).Muntean et. al. reported inhibitors to factor V following exposure tofibrin sealant during cardiac surgery and concluded that exposure totopical thrombin preparations may lead to the development of inhibitorsin the postoperative period that may cause bleeding complications (Ref.6). Based on the foregoing and other reports, Landsberg et, al.cautioned against use of bovine topical thrombin-based platelet gels inoral and maxilofacial procedures and expressed the need for alternativemethods of activating PRP in the oral surgery area (Ref 7).

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides an autologous platelet gel andbone graft matrix that is thrombin-free and promotes osteoconduction,osteoinduction and osteogenesis at a bone augmentation site, andinitiates and augments the bone replacement process. The implantcompositions of the present invention comprise an admixture ofplatelet-rich-plasma, osseoparticulates, a gelling initiator, and aosseoinductive carrier comprising a suspension of terminallydefribillated fibrillar collagen. The admixture is in-vitro cured toform a gelled composition that is formable for implantation into bonedeficient sites. Osseoparticluates in a variety of forms can be utilizedas the osseocondutive medium including autologous bone and marrow,allographic bone particulate, xenographic particulate bone substitutesand other calcium phosphate minerals. The collagen suspension is amicrofibrillar Type I collagen processed to undergo partial unwinding ofthe collagen stands at the ends thereof sufficient to maintainsuspension in a physiological medium and effective to act as a carrierfor the scaffolding and as an osseogenetic promoter.

[0010] REFERENCES. The publications set forth above are herebyincorporated by reference.

[0011] 1. “Platelet Gel: An autologous Alternative to Fibrin Glue withApplications in Oral and Maxilofacial Surgery”, Whitman, D H et al., J.Oral Maxilofacial Surgery, 1294-1299 (1997).

[0012] 2. “Platelet-rich plasma: Growth factor enhancement for bonegrafts”, Mars, RE et al., Oral Surg Oral Med Oral Pathol Oral RadiolEndod, Vol. 85, 638-646, (1998).

[0013] 3. “Anti-Bovine Thrombin Antibody”, Sosolik, R C et al.,Laboratory Medicine, Vol. 27, No. 10, 651-653 (1998).

[0014] 4. “Bovine thrombin-induced inhibitor of factor V and bleedingrisk in postoperative neurosurgical patients”, Spero J A, J Neurosurg,Vol 78, 817-820 (1993).

[0015] 5. “Redo cardiac surgery. Late bleeding complications tom topicalthrombin-induced factor V deficiency”, Cmolik, B L, J Thorac CardiovascSurg, Vol 105, 222-228 (1993).

[0016] 6. “Inhibitor to factor V after exposure to fibrin sealant duringcardiac surgery in a two-year-old child”, Muntean, W, Acta Paediar, Vol.83, 84-87 (1994).

[0017] 7. “Risks of using platelet-rich-plasma gel”, Landesberg, R, JOral Maxilofac Surg, Vol. 56, 1116-1117 (1998).

Description of the Preferred Embodiments

[0018] The autologous platelet gel and matrix of the present inventionis produced using patient derived platelet-rich-plasma fraction derivedfrom preoperative donated blood. For use as an autologous plateletsealant/bone graft matrix, the composition comprises by volume about 10to 60 parts of platelet-rich-plasma; 1 to 5 parts calcium chloride; 5 to60 parts of partially defibrillated Type I collagen; and 5 to 40 partsosseoparticulate. Such composition is made by: forming initially amixture of alkaline induced terminally frayed Type I fibrillar collagenand water; adjusting the mixture to a pH in the range of about 8 to 12;stirring said mixture at said range for a time sufficient to establish astable suspension; readjusting the suspension to a pH in thephysiological range; combining the suspension with calcium chloridesolution as an activator system; and combining the activator system withautologous platelet-rich-plasma under time and temperature conditionsyielding a clotted, formable gel, supplemented in accordance with theapplication by osseoparticulate.

[0019] The procedure for harvesting the platelet-rich-plasma may bepracticed in many variations, however, the procedure as described inMarx (Ref. 2) may be beneficially used herein. Such procedure involvesobtaining from the prospective implant patient, immediatelypreoperative, whole blood, which is transferred into a collectionreservoir containing a citrate- phosphate-dextose anticoagulant. Theanticoagulated whole blood is transferred to a centrifulge forseparation at about 5600 rpm into plural layers comprising:platelet-poor-plasma, upper layer: erythrocytes, lower layer; and the“buffy coat”, middle layer, containing the platelet-rich-plasmafraction. The platelet-poor-plasma, upper layer fraction, is removed byaspiration and the remainder recentrifuiged at 2400 rpm to furtherdefine the remaining fractions. The but coat middle layer containing theplatelet-rich-plasma is removed and stored at room temperature forfuture use. Such technique is reported to yield 500,000 to 1,000,000platelets in the PRP from a unit of whole blood.

[0020] While the foregoing separation procedure produces generallyacceptable results, the present invention has determined that greateryield and improved morphology can be obtained by more gentlysequestering the platelets. Herein, a sample of whole blood istransferred to a. Centrifuge tube containing a suitable coagulant suchas citrate solution. The tube is centrifuged in the range of 175 g to300 g for a period of time, 5 to 15 minutes, sufficient to delineate thesample into three distinct layers; a top layer containing platelet poorplasma, a buffy coat middle layer containing platelet-rich-plasma, and alower layer containing the red blood cells. Thereafter, the top andmiddle layers are transferred to a second Centrifuge tube. The tube iscentrifuged again at a gentle speed in the range of 200 g to 300 g for aperiod of time, generally 5 to 15 minutes, sufficient to delineateclearly the upper layer of platelet-poor-plasma and the now lower layercontaining the platelet-rich-plasma. The layers are thereafterseparated, and the platelet-rich-plasma reserved for use. Such gentlesequestration of the platelet-rich-plasma has been found to produceincreased yields of intact platelets exhibiting a morphologysubstantially unaffected by the separation process.

[0021] The gelling initiator or clotting activator is an inorganicsolution compatible with the graft constituents for effecting gelling orclotting of the autologous platelet gel and matrix. For thecollagen-based system of the present invention a calcium chlorideaqueous solution is preferred.

[0022] The carrier and osseoinductive constituent and second constituentof the activator is preferably a Type I fibrillar collagen derived fromallographic or xenographic sources. Type I Bovine collagen is preferred,however, other suitable animal sources such as mammalian or avian may beused. The collagen is alkaline treated to promote strand uncurling atthe ends of the fibers. Such processing permits the collagen to form astable aqueous suspension, Thereafter, the suspension is neutralized tophysiological conditions for use in the gel and platelet compositions ashereinafter described.

EXAMPLE 1 Preparation of Conventional Thrombin-Based Platelet Gel

[0023] Platelet-rich-plasma was obtained as set forth above, In a mixingbowl, 1 ml of platelet-rich-plasma, 60 nl of 91M calcium chloride, and10,000 U of Type 1 bovine thrombin were mixed to form a moldable viscousclot with the consistency of a gel.

EXAMPLE 2 Preparation of Collagen-Based Platelet Gel

[0024] In a mixing bowl, 1 ml of platelet-rich-plasma, 60 nl of 0.91Mcalcium chloride and .5 ml of collagen suspension (1 mg/ml) made inaccordance with example 3 below were thoroughly mixed and transferred toa heating surface maintained at 37° C. for about 10 minutes until aviscous gel was formed.

EXAMPLE 3 Preparation of Collagen Suspension

[0025] A collagen suspension was prepared by admixing .5 grams offibrillar Type I Bovine Collagen (J&J Medical Systems, Product No 1984)in 5 ml of distilled water. The pH of the mixture was increased to 10.2with the addition of 10M Sodium Hydroxide. The adjusted mixture wasgently stirred for a period of 8 hours at which time the collagenremained in stable suspension. The adjusted suspension was neutralizedto physiologic conditions at a pH of 7.2 by the addition or 10Mhydrochloric acid. Microscopic examination of the collagen fibersindicated frayed termini on the fiber ends resultant from aboveprocessing.

EXAMPLE 4 Determination of PDGF and TGF-B Content

[0026] In order to determine the effects of the thrombin-based gel asprepared in accordance with Example 1 and the collagen-based gel of thepresent invention as prepared in accordance with Example 2 in therelease of Platelet Derived Growth Factor (PDGF) and Transforming GrowthFactor-Beta (TGF-B), 10 ml samples based on different blood sources wereanalyzed in accordance with established protocols

[0027] The results are set forth below in Table A TABLE A Sample A B CPDGF (Units) Thrombin 157.3 113.7 107.4 Collagen 155.4 127.7 130.3 TGF-B(Units) Thrombin 118.0 123.9 154.7 Collagen 111.9 130.9 169.21

[0028] The foregoing demonstrates that both gel preparations aresubstantially equally effective in releasing the noted growth factorsassociated with bone and tissue augmentation.

EXAMPLE 5 Gel Conditions

[0029] To illustrate the effect of time, temperature and calciumchloride concentration on clotting time and consistency three samples ofgels in accordance with Example 2 above were prepared with theexceptions that two samples used the 10% calcium chloride and one samplean equal volume of 2M solution. The 10% samples clotted at roomtemperature in 11.5 minutes and at 37° C. in 4 and 4.5 minutes. The 2Msolution did not clot at either temperature.

EXAMPLE 6 Clot Consistency

[0030] To illustrate the effect of calcium chloride concentration on thecollagen-based platelet gel, three samples of the gel were prepared inaccordance with Example 2 in the following proportions; Sample A B C PRP100 ml 100 ml 100 ml Calcium Chloride 14 ml 50 ml 6 ml Collagen 20 ml 50ml 100 ml Clotting Time no clot no clot 7.6 min

[0031] Such conditions indicated that increased amounts of the calciumchloride adversely affected the desired clotting characteristics, evenin the presence of temperature activation.

EXAMPLE 7 Preparation of Platelet Gel for Implantation

[0032] The collagen suspension prepared in Example 5, 10 ml was mixed ina sterile glass bowl with 10 ml of 0.91M calcium chloride. The collagenmixture was then mixed with 50 ml. of plasma rich platelets contained ina second glass container and mixed until evenly distributed. Preliminarygelling was noted. The glass container was placed on a hot platemaintained at 37° C. for clotting. After 30 minutes of heating, theresultant platelet gel was removed and found to be uniformly clotted andreadily moldable into retentive shapes conformal to all implant site.

EXAMPLE 8 Preparation Platelet Augmentation Osteograph Composition

[0033] Prior to heating, 25 mg. of cadavillar particulate was added tothe ungelled mixture and mixed until well incorporated. The resultantmixture in the glass container was placed on a hot plate maintained at37° C. for gellation. After 30 minutes of heating, platelet gel withparticulate was removed and bound to be readily moldable into typicaldental implant shapes.

EXAMPLE 9 Implantation of Osteogrph Composition.

[0034] A quantity of composition prepared in accordance with Example 8was preliminary molded and inserted into a mandibular void of a subject.The composition was further defined to desired shape. The surroundingtissue was then closed by sutures. Visual inspection during thepostoperative period did not indicate any inflammation or swellingattributable to the implant. Subsequent visual and radiologicalobservation indicated progressive increase in both load-bearing andcomplete osteointegration in accordance with conventional analysis. Noallergic or antibody reaction was noted for a period of up to 4 months.At the end of 5 months, the implant was fully integrated and loadsupporting.

[0035] The bone graft material of the present invention has applicationin craniofacial reconstruction, periodontal defects, jointreconstruction, fracture repair, orthopedic surgical procedures, spinalfusion, bone defects, odontolological defects inosteoconductive/osteoinductive grafting applications.

[0036] The gel matrices of the present invention also have applicationsin any surgical or invasive technique in which manipulative or promotionof wound tissue deficit healing is intended.

[0037] The gels and matrices of the present invention also haveapplication as matrices for the storage and encapsulation of cellularmoieties such as pancreatic islets, xenographic or allographic,hepatocytic cells and the like.

[0038] Various modifications of the invention in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description. Such modifications are intended to be withinthe scope of the appended claims.

What is claimed:
 1. An autologous gel, comprising; an aqueous suspensionof Type I fibrillar collagen alkaline treated to created partiallyfrayed termini; calcium chloride; and platelet-rich-plasma processed toform an in vitro formable matrix.
 2. A method of making an implant gel,comprising the steps of: making (( an admixture of Type I collagen inphysiological suspension, an aqueous ( solution of calcium chloride andbone particulate; and adding to said admixture platelet-rich-plasma inan amount sufficient to establish a viscous formable gel.
 3. The methodas recited in claim 2 including the step of heating said gel at aroundbody temperature to accelerate formation of the gel.
 4. In aplatelet-based sealant characterized by a mixture of calcium chloride,thrombin and a source of platelets, the improvement comprising:substituting an effective amount of terminally frayed Type Imicrofibrillar collagen for said thrombin.
 5. A cellular matrix forcellular moieties, comprising by volume 10 to 60 parts ofplatelet-rich-plasma; 1 to 5 parts calcium chloride; 5 to 60 parts ofpartially defibrillated Type I collagen; and 5 to 40 parts boneparticulate.
 6. A method of making an in-vitro thrombic agent for use inplatelet-rich-plasma compositions comprising the steps of: forming amixture of Type I fibrillar collagen and water; adjusting the mixture toa pH in the range of about 8 to 12; stirring said mixture at said rangefor a sufficient time to establish a stable suspension; and readjustingthe suspension to a pH in the physiological range.
 7. The method asrecited in claim 7 wherein said mixture has a pH in the range of 9.5 to110 and said physiological range is between 6.8 and 7.2.
 8. A method ofseparating from whole blood a predominantly platelet-rich-plasmafraction comprising the steps of: centrifuging a volume of whole bloodat a first separating condition at centrifugal forces in the range ofabout 150 g to 400 g and a time sufficient to differentiate a top layer,a middle layer and a bottom layer; separating said top layer and saidmiddle layer from said bottom layer; recentrifuging the top layer andthe middle layer at a second separating condition at centrifugal forcesin the range of about 225 g to 300 g for a time to differentiate furthersaid top layer from said middle layer; and separating said middle layerfrom said top layer.
 9. The method as recited in claim 9 wherein saidcentrifugal forces in at said first separating condition are in therange of about 175 g to 250 g.
 10. The method as recited in claim 9wherein said centrifugal forces at said second separating condition arein the range of about 225 g to 275 g.
 11. A cellular matrix comprising:a cellular moiety in admixture with an autologous platelet-based gelincluding autologous platelet-rich-plasma, terminally frayedmicrofibrillar Type I collagen, and a gelling initiator.
 12. The matrixas recited in claim 11 wherein said cellular moiety is pancreaticislets.